The long-term objective of this application is thorough and intensive training in molecular biology that will prepare Dr. Glenn Gerhard for future independent basic research into molecular mechanisms of aging. Phase I includes academic training through graduate courses, seminars, presentation, and teaching, with emphasis on gene expression and development. An advisory committee will serve to monitor Dr. Gerhard's progress and supplement the research training. The didactic program is built around a research project in the laboratories of Drs. Nancy Cooke and Stephen Liebhaber, Departments of Medicine and Human Genetics. The specific research project will focus on disruption of the vitamin D binding protein (DBP/GC globulin) gene by gene targeting through homologous recombination in embryonic stem (ES) cells. DBP is the oldest member of a multigene family which includes albumin (ALB) and alpha-fetoprotein (AFP), whose diverse functions include transport of vitamin D sterols, high affinity binding of actin monomers, and a role in neutrophil chemotaxis. A deletion of DBP has not been detected despite extensive screening suggesting DBP functions are critical to normal development.
The specific aims for Phase I research include cloning and characterizing a portion of the mouse DBP gene, construction of a replacement targeting vector, and the generation of DBP-/DBP+ ES cells. The AFP gene will be targeted in parallel studies. To do this, Dr. Gerhard will first clone and characterize a portion of the mouse DBP gene containing exons 2 and 3. He will construct a targeting vector for use in positive-negative drug selection by insertion of the E. coli neomycin resistance gene into an exon of the cloned mouse DBP genomic fragment. The Herpes Simplex Virus thymidine kinase gene will then be ligated to one end of the genomic fragment. ES cells will be electroporated with this construct and doubly selected with G418 and gancyclovir. Dual drug resistant colonies, reflecting integration of the neo gene and loss of the thymidine kinase gene, will be selected and analyzed by PCR and Southern blotting for successfully targeted homologous recombinant DBP-/DBP+ ES cells. During the phase II research plan, the targeted ES cells will be microinjected into C57B1/6J blastocysts and reimplanted into pseudopregnant recipients to generate chimeras. Male chimeras will then be back-crossed to C57B1/6J females to assess germ-line transmission. Germ-line progeny of the back-crosses will be obligate DBP-/DBP+ mice. Intercrossing these mice will produce DBP-/DBP- homozygotes. Depending upon phenotype, extensive functional analysis of DBP-/DBP- mice will include evaluation of vitamin D metabolism, actin binding studies, and neutrophil chemotaxis assays. Site-directed mutagenesis of the DBP gene through transgenic techniques and/or further gene targeting is also planned.